A rare clinical presentation of human Dirofilaria repens infection as a pseudo-tumour of the epididymis - Case Report.

Introduction. Epididymal dirofilariasis is one of the unusual manifestations of this zoonosis. In Slovakia, this is the third case out of 20 Dirofilaria repens infected patients in whom the worm (the parasite) was identified in the epididymis. The patient felt a painless tumour about 2 cm in size on the left testicle. During ultrasound examination, the radiologist observed a cyst containing a live worm within the epididymis. After mechanical stimulation, the movement characteristic for filarial worms (´filarial dance sign´) appeared. An orchiectomy was performed at the Department of Urology in the University Hospital in Kosice. Histopathology confirmed a parasitic cyst with a worm, and based on characteristic morphological features, the parasite was identified as Dirofilaria repens. Objective. The aim of case report is to alert physicians to the possibility dirofilarial infection of the epididymis, where tumors and cystic structures often occur and to present clinical signs of the disease.

The application of proteomic methods (MALDI-toff MS) for studying protein profiles of some nematodes (dirofilaria and ascaris) for differentiating species.

INTRODUCTION: Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-toff MS) is a reliable method for diagnosing a number of bacterial and fungal infections. It is also effective as a method of rapid diagnosis of several parasitic agents. We used MALDI-toff MS to study the protein profiles of four nematodes: Dirofilaria repens, Dirofilaria. immitis, Ascaris suum and Ascaris lumbricoides. METHODS: We studied the protein profiles of dirofilaria (five of each species: D. repens and D. immitis) and ascaris (five of each species: A. suum and A. lumbricoides), using a proteomic analysis based on MALDI-toff MS. RESULTS: Analysis of protein extracts of dirofilaria and ascaris showed spectra with high-intensity peaks in the range of 2-20 kDa. The quality of the spectra (clear graphical reflection of mass/charge to luminous intensity, consistent in repeated analyzes) and the intensity of the spectral peaks were consistent in all samples of the same species. The spectra profiles of D. repens and D. immitis differed in eight major peaks which makes it possible to differentiate species according to the protein profile. The spectra profiles obtained from A. suum and A. lumbricoides proteins differed slightly in 3 major peaks in both species and were discovered in m/z 13000; 13400 and 14400. The protein peaks in diapason 3000 kD-7300 kD specific for all genus ascaris are constant. CONCLUSIONS: MALDI-toff MS-based proteomic analysis can serve as an effective taxonomic tool for parasitological studies.

Whether Dirofilaria repens parasites from South India belong to zoonotic Candidatus Dirofilaria hongkongensis (Dirofilaria sp. hongkongensis)?

The canine and zoonotic dirofilarioses are arthropod-borne parasitic infections caused by nematodes of the genus Dirofilaria, infecting canines, felines and humans throughout the world. Dirofilaria repens was considered as the most common cause of human dirofilariosis in Kerala. In the present study, molecular characterization of Dirofilaria isolates causing dirofilariosis in humans, dogs and jackal from Kerala, South India was undertaken by performing sequence and phylogenetic analysis based on cytochrome oxidase subunit I (COI) gene. The live worms from swellings/ nodules in subconjunctiva or subcutaneous tissue or scrotum were recovered from humans (n = 3), dogs (n = 4) and one jackal. The PCRs targeting a repetitive fragment, 18S rRNA and COI genes yielded products of ~246 bp, ~875 bp and ~350 bp respectively in all the samples. The sequence analysis of 18S rRNA gene revealed the closest identity (98 to 99%) with an already published sequence of D. repens isolated from a human in Japan. However, based on the sequence and phylogenetic analysis of partial sequences of COI gene, the Dirofilaria infecting both animals (dogs, jackal) and humans native to Kerala, South India were identified as genetically conserved and closely related to Dirofilaria sp. hongkongensis. Hence, the results of the present study suggested the existence of Candidatus Dirofilaria hongkongensis (Dirofilaria sp. hongkongensis) in Kerala, South India causing zoonotic filariosis in canines and humans.

Recent advances on Dirofilaria repens in dogs and humans in Europe.

Dirofilaria repens is a nematode affecting domestic and wild canids, transmitted by several species of mosquitoes. It usually causes a non-pathogenic subcutaneous infection in dogs and is the principal agent of human dirofilariosis in the Old World. In the last decades, D. repens has increased in prevalence in areas where it has already been reported and its distribution range has expanded into new areas of Europe, representing a paradigmatic example of an emergent pathogen. Despite its emergence and zoonotic impact, D. repens has received less attention by scientists compared to Dirofilaria immitis. In this review we report the recent advances of D. repens infection in dogs and humans, and transmission by vectors, and discuss possible factors that influence the spread and increase of this zoonotic parasite in Europe. There is evidence that D. repens has spread faster than D. immitis from the endemic areas of southern Europe to northern Europe. Climate change affecting mosquito vectors and the facilitation of pet travel seem to have contributed to this expansion; however, in the authors' opinion, the major factor is likely the rate of undiagnosed dogs continuing to perpetuate the life-cycle of D. repens. Many infected dogs remain undetected due to the subclinical nature of the disease, the lack of rapid and reliable diagnostic tools and the poor knowledge and still low awareness of D. repens in non-endemic areas. Improved diagnostic tools are warranted to bring D. repens diagnosis to the state of D. immitis diagnosis, as well as improved screening of imported dogs and promotion of preventative measures among veterinarians and dog owners. For vector-borne diseases involving pets, veterinarians play a significant role in prevention and should be more aware of their responsibility in reducing the impact of the zoonotic agents. In addition, they should enhance multisectorial collaboration with medical entomologists and the public health experts, under the concept and the actions of One Health-One Medicine.

Filarial nematode (order: Spirurida) surveillance in urban habitats, in the city of Pécs (Hungary).

As part of the seasonal mosquito control activities in the city of Pécs (Baranya County, Hungary), a total of 1123 adult female mosquitoes belonging to 18 species (including the invasive species Aedes koreicus) were collected from human-inhabited areas, using CO2-baited traps, during two consecutive years. To survey the presence and prevalence of filarial parasites in these mosquitoes, we performed a molecular survey for filarial DNA, attempted by PCR using generic primers (COI), and followed by DNA sequencing. Filaroid nematode DNA was detected in 4% of investigated mosquito pools. Out of 410 pools, 9 pools of mosquitoes were positive for Dirofilaria repens (Aedes vexans, Aedes koreicus, Coquillettidia richiardii), and/or Dirofilaria immitis (Ae. vexans, Cq. richiardii), and further 8 pools were positive for Setaria tundra (Ae. vexans, Cq. richiardii). Our study provides novel insight for prevalence of filaroid nematodes in mosquitoes occurring in close proximity to humans, thereby highlights the possible human and veterinary health importance of these mosquito species, including the recently introduced invasive mosquito Ae. koreicus.

Circulation of Dirofilaria repens and Dirofilaria immitis in Moldova.

BACKGROUND: Over the last two decades, a significant spread of dirofilariasis has been observed in eastern and central Europe. However, data on the circulation of Dirofilaria spp. in Moldova were absent although direct neighbor states reported high incidence rates of human dirofilariasis. METHODS: Daily mean temperature data were used to calculate Dirofilaria spp. development units, which were used to estimate the potential for complete extrinsic development in the mosquitoes (= sum of potential Dirofilaria spp. transmission days). In addition, 4,481 adult female mosquitoes were collected from 25 trapping sites. From 2010 to 2015, sampling was conducted with Centers for Disease Control miniature light traps, indoor resting mosquito collections as well as human landing catches in urban, rural and natural areas. Mosquitoes were analyzed for the presence of D. repens and D. immitis DNA using a duplex real-time PCR assay targeting nucleotide differences within the cytochrome c oxidase subunit 1 (D. repens) and 16S rRNA gene fragment (D. immitis). RESULTS: The average of the yearly sum of potential Dirofilaria spp. transmission days between 2010 and 2015 ranged from 90 to 140 days with an increasing gradient from the North to the South of Moldova. Positive mosquito pools for D. repens were found countrywide at 13 of the 25 trapping sites and in 17 of the 22 screened mosquito taxa (26.51% of all 347 tested pools), while D. immitis was detected only at 4 of the trapping sites (Center and South) in 4 different mosquito species (8.65% of all 347 tested pools). Highest infection rates (EIR) per 100 specimens for both Dirofilaria species were found in An. maculipennis (s.l.) (D. repens: EIR = 4.91; D. immitis: EIR = 2.01), whereas the most frequent mosquito taxon Cx. pipiens (s.l.)/torrentium had significantly lower infections rates (D. repens: EIR = 0.88; D. immitis: EIR = 0.47). CONCLUSIONS: The temperature conditions in Moldova are suitable for transmission of Dirofilaria spp. within the entire country, which is supported by a wide distribution of Dirofilaria spp.-positive mosquitoes with high infection rates. The low number of reported human cases most likely does not reflect the current epidemiological situation of dirofilariasis in Moldova.

First molecular identification of Dirofilaria spp. (Onchocercidae) in mosquitoes from Serbia.

Dirofilariosis is a common and widespread veterinary health issue in several European countries with notable zoonotic potential. The causative agents are Dirofilaria immitis and Dirofilaria repens nematoda species which are transmitted by different mosquito vectors. Similar to other mosquito-borne infections, the knowledge about mosquito species involved in disease transmission is crucial for the complex understanding of local transmission cycles. Since there is no available data on mosquito species, potentially involved in disease transmission from Serbia, 6369 female mosquito individuals were retrospectively tested for Dirofilaria nematodes, collected from 13 localities in Vojvodina province, Serbia, in 2013. Altogether, 8.33 % of tested pools showed positivity, composed of five mosquito species, mainly, Culex pipiens and Aedes vexans. D. immitis and D. repens were both detected from multiple localities, during the whole period of mosquito breeding season, which provides the first data on local transmission characteristics regarding mosquitoes from the Balkans.

A Rare Human Case of Dirofilaria repens Infection in the Subcutaneous Posterior Thorax with Molecular Identification.

The emergence of Dirofilarial infections in Asia including Vietnam is a clinically significant threat to the community. We here report a rare case of subcutaneous Dirofilaria repens infection on the posterior thoracic wall in a young woman presenting a painful, itchy, and palpable nodule. The adult worm was identified by mitochondrial cox1 and nuclear ITS-2 sequence determination. The diagnosis was additionally confirmed by 16S rRNA sequencing of the endosymbiont Wolbachia pipientis commonly co-existing with D. repens. This is a rare case of subcutaneous human infection on the posterior thoracic region caused by D. repens.

A Rare Human Case of Dirofilaria repens Infection in the Subcutaneous Posterior Thorax with Molecular Identification

The emergence of Dirofilarial infections in Asia including Vietnam is a clinically significant threat to the community. We here report a rare case of subcutaneous Dirofilaria repens infection on the posterior thoracic wall in a young woman presenting a painful, itchy, and palpable nodule. The adult worm was identified by mitochondrial cox1 and nuclear ITS-2 sequence determination. The diagnosis was additionally confirmed by 16S rRNA sequencing of the endosymbiont Wolbachia pipientis commonly co-existing with D. repens. This is a rare case of subcutaneous human infection on the posterior thoracic region caused by D. repens.

Dirofilaria repens Railliet et Henry, 1911--a new parasite acclimatized in Poland.

Dirofilariosis caused by the Dirofilaria repens nematodes is widely dispersed in southern Europe, Asia and Africa among dogs, cats, other carnivores and occasionally, humans. The first case of D. repens infection, found in Poland in 2007, concerned humans. In 2009, dirofilariosis was first registered in dogs in 3 focuses in central Poland, Warsaw, Pruszków and Zyrardów, whose range grew considerably with the subsequent identification of D. repens microfilariae in 119 dogs in Warsaw and 18 districts of the Mazowieckie Province. The microfilariae of D. repens were found in blood samples taken from 1588 dogs from all 16 provinces of Poland. D. repens was also recently detected in a mixture of Culex pipiens and Aedes vexans mosquitoes collected in Mazowieckie Province using Real Time PCR. The results of this study confirms the acclimatisation of D. repens on the territory of Poland and a possibility for it to close its life cycle in domestic species of mosquito.

Adult of Dirofilaria repens in a dog with recurrent multiple subcutaneous nodular lesions.

Subcutaneous dirofilariosis is a zoonosis parasitizing the dog, transmitted by Culicidae mosquitoes. An increasing number of new cases have been reported both in dogs and in humans all over the world. The aim of this work was to describe an uncommon case of recurrent multiple subcutaneous nodules by Dirofilaria repens in a 2-year-old dog. Surgical excision of two nodules on both hind limbs revealed a single thread-like live nematode and multiple parasite fragments, respectively. The parasite was morphologically identified as an adult female of D. repens. Haematoxylin-eosin tissue sections of nodules revealed a diffuse pyogranulomatous angiocentric dermatitis with an intense eosinophilic infiltrate throughout the dermis. One month post-surgery, a new nodular lesion was detected on the right thigh and resolved spontaneously in a few weeks. During the following 3 months, two other nodules were briefly noted both on the right hip and on the left side of the chest. The dog was periodically treated with milbemycin oxime and praziquantel, starting a week before the appearance of the first nodule. Microfilariae were not detected neither in the bloodstream nor in fine-needle aspirates at any time, and the diagnosis was made only after surgery. Practitioners should consider dirofilariosis in the differential diagnosis of subcutaneous swellings, especially if they have a recurrent behaviour, previously described in humans but not in dogs.

Histological and molecular confirmation of the fourth human case caused by Dirofilaria repens in a new endemic region of Slovakia.

Dirofilariosis is considered to be the arthropod vector-borne disease with the fastest spread in Europe. Slovakia belongs to new endemic regions for canine and human infections. This paper reports the fourth human case, where diagnosis was confirmed using computed tomography (CT), histological and molecular examinations - for the first time in this endemic region. The epidemiological history of this case indicated the autochthonous origin, and infection manifested by intense swelling in the periocular region, where a subcutaneous nodule was localized. Microscopic analysis of cross-sections of the surgically removed nodule confirmed the presence of a single male worm of Dirofilaria repens, indicated by the external cuticular ridges, the robust muscle cells and a single male sex organ seen as one tube beside the intestine. Considering that the worm morphology was partially damaged, molecular study was performed using DNA isolated from formalin-fixed and paraffin-embedded tissue sections of the worm. In polymerase chain reactions (PCR) a set of primers specific for D. repens DNA for the CO1-encoding gene amplified the expected 246 bp product using a high concentration of DNA template. Our diagnostic approach, involving molecular techniques, showed that identification of D. repens from excised tissues is possible even when the morphology of the worm and DNA are damaged during tissue processing. It is expected that the spread of this disease will continue due to climatic changes in central Europe. This warrants higher awareness among clinicians, who will initially be approached by patients, and better co-operation with parasitologists in newly endemic countries.

Differentiation of the microfilariae of Dirofilaria immitis and Dirofilaria repens in stained blood films.

An examination was made of Giemsa-stained microfilariae in thin blood films from (n = 9) dogs naturally or experimentally infected with Dirofilaria immitis or Dirofilaria repens. Morphological measurements (total length, length of cephalic space, anterior end to nerve ring and last body nucleus, and nucleus-free tail tip) were made on 2-6 microfilariae from each dog with the use of digitally captured images and imaging software. The microfilariae of D. repens were significantly greater (P < 0.001) in all measured dimensions except for the length of the cephalic space, which was significantly shorter (P < 0.001) than that of D. immitis. The cephalic space of D. repens was characterized by being short and routinely being terminated by a distinct pair of nuclei that were separate from the remaining somatic nuclei of the microfilaria. The cephalic space of the smaller microfilaria of D. immitis was longer and did not have the distinct nuclei separated from the somatic column nuclei near the anterior end. The character of the cephalic space seems to be a criterion that could be routinely used for the easy differentiation of these 2 microfilariae in stained blood films.